Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

BACKGROUND: Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. METHODS: In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. RESULTS: In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. CONCLUSIONS: Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

Polyphenol Extracts from Ziziphus jujuba Mill. "Junzao" Attenuates Ulcerative Colitis by Inhibiting the NLRP3 and MAPKs Signaling Pathways and Regulating Gut Microbiota Homeostasis in Mice.

SCOPE: Polyphenols are the major active substances in red jujube fruit, and their anti-inflammatory and antioxidant activities suggest their potential utility in the prevention of ulcerative colitis (UC). METHODS AND RESULTS: In this study, the effect of polyphenol extracts from red jujube (Ziziphus jujuba Mill. "Junzao") (PERJ) on the dextron sulfate sodium (DSS)-induced UC mice is investigated. The result shows that PERJ effectively improves clinical symptoms, including food and water intake, the disease activity insex (DAI) and spleen index, and routine blood levels, and alleviates the shortening of the colon, in mice with DSS-induced UC. Meanwhile, PERJ remarkably decreases the expression of proinflammatory factors. Moreover, PERJ repairs intestinal barrier damage by increasing the expression level of mucin 2 and mucin 3, and the result is also confirmed in the histological assessment. Besides, the expression levels of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and mitogen-activated protein kinase cascade (MAPKs) signaling pathway-related proteins are inhibited by the PERJ administration. Finally, 16S rRNA sequencing analyses reveal that PERJ reverses intestinal microbiota dysbiosis by enhancing the abundance of Firmicutes and decreasing that of Proteobacteria and Bacteroidetes. CONCLUSION: PERJ probably inhibits the development of UC by suppressing the NLRP3 and MAPKs signaling pathways and regulating gut microbiota homeostasis, and can be considered as a potential resource for preventing UC.

α-Hederin induces paraptosis by targeting GPCRs to activate Ca2+/MAPK signaling pathway in colorectal cancer.

BACKGROUND: Non-apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α-hederin (α-hed) induces caspase-independent paraptotic modes of cell death. PURPOSE: The present study is aimed to investigate the role of α-hed induces paraptosis and the associated mechanism of it. METHODS: The cell proliferation was detected by CCK-8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α-hed. Molecular docking methods were used to evaluate binding abilities of α-hed with targets. The expressions of genes and proteins were analyzed by RT-qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo. RESULTS: α-hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg-2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α-hed. Besides, both Swiss prediction and molecular docking showed that the structure of α-hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)-ß3/ inositol 1,4,5-trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCß3, IP3R, and PKCα were increased by α-hed. After blocking the GPCR signaling, α-hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α-hed activated MAPK cascade by elevating Ca2+ flux. Since non-apoptotic cell death is presently emerging as a potential direction to overcome chemo-drug resistance, we then found α-hed also induced paraptosis in 5-fluorouracil-resistant (5-FU-R) CRC cells, and it reduced the growth of 5-FU-R CRC xenografts. CONCLUSIONS: Collectively, our findings proved α-hed as a promising candidate for inducing non-apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic-based chemo-resistance in CRC.

Trauma-associated extracellular histones mediate inflammation via a MYD88-IRAK1-ERK signaling axis and induce lytic cell death in human adipocytes.

Despite advances in the treatment and care of severe physical injuries, trauma remains one of the main reasons for disability-adjusted life years worldwide. Trauma patients often suffer from disturbances in energy utilization and metabolic dysfunction, including hyperglycemia and increased insulin resistance. White adipose tissue plays an essential role in the regulation of energy homeostasis and is frequently implicated in traumatic injury due to its ubiquitous body distribution but remains poorly studied. Initial triggers of the trauma response are mainly damage-associated molecular patterns (DAMPs) such as histones. We hypothesized that DAMP-induced adipose tissue inflammation contributes to metabolic dysfunction in trauma patients. Therefore, we investigated whether histone release during traumatic injury affects adipose tissue. Making use of a murine polytrauma model with hemorrhagic shock, we found increased serum levels of histones accompanied by an inflammatory response in white adipose tissue. In vitro, extracellular histones induced an inflammatory response in human adipocytes. On the molecular level, this inflammatory response was mediated via a MYD88-IRAK1-ERK signaling axis as demonstrated by pharmacological and genetic inhibition. Histones also induced lytic cell death executed independently of caspases and RIPK1 activity. Importantly, we detected increased histone levels in the bloodstream of patients after polytrauma. Such patients might benefit from a therapy consisting of activated protein C and the FDA-approved ERK inhibitor trametinib, as this combination effectively prevented histone-mediated effects on both, inflammatory gene activation and cell death in adipocytes. Preventing adipose tissue inflammation and adipocyte death in patients with polytrauma could help minimize posttraumatic metabolic dysfunction.

l-Theanine Prevents Colonic Damage via NF-κB/MAPK Signaling Pathways Induced by a High-Fat Diet in Rats.

SCOPE: l-Theanine (l-Thea) is an amino acid which is naturally present in tea leaves. It has been associated with possible health advantages, including obesity prevention, but the underlying molecular mechanisms have not been elucidated. METHODS AND RESULTS: A multiomics approach is utilized to examine the mechanism by which l-Thea exerts its antiobesity effects. This study reveals that l-Thea administration significantly ameliorates high-fat diet (HFD)-induced obesity in rats by improving body weight and hyperlipidemia. l-Thea mitigates HFD-induced inflammation and reverses hepatic and colonic damage, and intestinal barrier. This research verifies that the preventive effect of l-Thea on obesity in rats induced by an HFD with colitis is accomplished by suppressing the phosphorylation of important proteins in the NF-κB/mitogen-activated protein kinase (MAPK) pathways. Metabolome analysis reveals that l-Thea regulates HFD-induced metabolic disorders, specifically through modulation of steroid hormone biosynthesis. Microbiome analysis reveals that l-Thea mitigates HFD-induced dysbiosis by increasing the relative abundance of obesity-associated probiotic bacteria, including Blautia coccoides and Lactobacillus murinus, while simultaneously suppressing the abundance of pathogenic bacteria. CONCLUSIONS: l-Thea alleviates colitis generated by an HFD by restoring the integrity of the intestinal barrier, suppressing inflammation through regulation of MAPK/NF-κB signaling pathways, and enhancing microbial metabolism in colon.

Huanglian-banxia promotes gastric motility of diabetic rats by modulating brain-gut neurotransmitters through MAPK signaling pathway.

BACKGROUND: Gastric motility disorder is an increasingly common problem among people with diabetes. Neurotransmitters have been recognized as critical regulators in the process of gastric motility. Previous study has shown that herb pair huanglian-banxia (HL-BX) can improve gastric motility, but the underlying mechanism is still unclear. The aim of this study was to further investigate the role of HL-BX in modulating brain-gut neurotransmission to promote gastric motility in diabetic rats, and to explore its possible mechanism. METHODS: The diabetic rats were divided into five groups. Gastric emptying rate, intestinal propulsion rate, body weight, and average food intake were determined. Substance P (SP), 5- hydroxytryptamine (5-HT), and glucagon-like peptide -1 (GLP-1) in the serum were measured by enzyme-linked immunosorbent assay. Dopamine (DA) and norepinephrine (NE) in the brain were analyzed by high-pressure liquid chromatography with a fluorescence detector. Protein expression of the tissues in the stomach and brain was determined by Western blot. KEY RESULTS: HL-BX reduced average food intake significantly, increased body weight, and improved gastric emptying rate and intestinal propulsion rate. HL-BX administration caused a significant increase in SP, GLP-1, and 5-HT, but a significant decrease in DA and NE. Interestingly, HL-BX regulated simultaneously the different expressions of MAPK and its downstream p70S6K/S6 signaling pathway in the stomach and brain. Moreover, berberine exhibited a similar effect to HL-BX. CONCLUSIONS: These results indicated that HL-BX promoted gastric motility by regulating brain-gut neurotransmitters through the MAPK signaling pathway. HL-BX and MAPK provide a potential therapeutic option for the treatment of gastroparesis.

Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

Protein interaction network analysis of mTOR signaling reveals modular organization.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (phosphoinositide 3-kinases), protein kinases (AKT), and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex coimmunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after 1 h, despite phosphorylation changes observed after only 5 min. Using small molecule inhibitors of phosphoinositide 3-kinase, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed "modules", that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to the activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

Cyclovirobuxine D inhibits growth and progression of non­small cell lung cancer cells by suppressing the KIF11­CDC25C­CDK1­CyclinB1 G2/M phase transition regulatory network and the NFκB/JNK signaling pathway.

Lung cancer is the leading cause of cancer­related mortality worldwide. Non­small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer and is associated with low 5­year overall survival rates. Therefore, novel and effective chemotherapeutic drugs are urgently required for improving the survival outcomes of patients with lung cancer. Cyclovirobuxine D (CVB­D) is a natural steroidal alkaloid, used for the treatment of cardiovascular diseases in Traditional Chinese Medicine. Several studies have also demonstrated the antitumor effects of CVB­D. Therefore, in the present study, the therapeutic effects of CVB­D in lung cancer and the underlying mechanisms were investigated using the in vivo xenograft model of NSCLC in nude mice and in vitro experiments with the NSCLC cell lines. Bioinformatics analyses of RNA­sequencing data, and cell­based functional assays demonstrated that CVB­D treatment significantly inhibited in vitro and in vivo NSCLC cell proliferation, survival, invasion, migration, angiogenesis, epithelial­to­mesenchymal transition and G2/M phase cell cycle. CVB­D exerted its antitumor effects by inhibiting the KIF11­CDK1­CDC25C­cyclinB1 G2/M phase transition regulatory oncogenic network and the NF­κB/JNK signaling pathway. CVB­D treatment significantly reduced the sizes and weights and malignancy of xenograft NSCLC tumors in the nude mice. In conclusion, the present study demonstrated that CVB­D inhibited the growth and progression of NSCLC cells by inhibiting the KIF11­CDK1­CDC25C­CyclinB1 G2/M phase transition regulatory network and the NF­κB/JNK signaling pathway. Therefore, CVB­D is a promising drug for the treatment of NSCLC patients.

Active compounds and potential targets of Shuganning injection in the treatment of hepatocellular carcinoma by network pharmacology and in vitro validation.

Shuganning injection (SGNI), a TCM (traditional Chinese medicine) injection with good hepatoprotective effects, exerted therapeutic effects on hepatocellular carcinoma (HCC). However, the active compounds and effects of SGNI on HCC remain unclear. The objective of this study was to investigate the active compounds and potential targets of SGNI in the treatment of HCC, and explore the molecular mechanisms of main compounds. Network pharmacology was applied to predict the active compounds and targets of SGNI on cancer. The interactions between active compounds and target proteins were validated by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and pull-down assay. The in vitro test of the effects and mechanism of vanillin and baicalein was elucidated by MTT, western blot, immunofluorescence, and apoptosis analysis. According to compound characteristics, targets, etc., two typical active ingredients (vanillin and baicalein) were selected as representatives to explore the effects on HCC. Vanillin (an important food additive) bound to NF-κB1 and baicalein (a bioactive flavonoid) bound to FLT3 (FMS-like tyrosine kinase 3) were confirmed in this study. Vanillin and baicalein both inhibited cell viability and promoted apoptosis of Hep3B and Huh7 cells. In addition, both vanillin and baicalein could enhance the activation of the p38/MAPK (mitogen-activated protein kinase) signaling pathway, which may partially explain the anti-apoptosis effects of the two compounds. In conclusion, two active compounds of SGNI, vanillin and baicalein, promoted apoptosis of HCC cells via binding with NF-κB1 or FLT3, and regulating the p38/MAPK pathway. Baicalein and vanillin may be good candidates for HCC treatment on drug development.

Royal jelly protects brain tissue against fluoride-induced damage by activating Bcl-2/NF-κB/caspase-3/caspase-6/Bax and Erk signaling pathways in rats.

This study is aimed at determining whether royal jelly (RJ) which has a powerful antioxidant property prevents fluoride-induced brain tissue damage and exploring whether Bcl-2/NF-κB/ and caspase-3/caspase-6/Bax/Erk pathways play a critical role in the neuroprotective effect of RJ. Wistar albino rats were chosen for the study, and they were randomly distributed into six groups: (i) control; (ii) royal jelly; (iii) fluoride-50; (iv) fluoride-100; (v) fluoride-50 + royal jelly; (vi) fluoride-100 + royal jelly. We established fluoride-induced brain tissue damage with 8-week-old male Wistar albino rats by administration of fluoride exposure (either 50 mg/kg or 100 mg/kg bw) through drinking water for 8 weeks. Then, the study duration is for 56 days where the rats were treated with or without RJ (100 mg/kg bw) through oral gavage. The effects of RJ on glutathione (GSH), catalase activity (CAT), and malondialdehyde (MDA) levels were determined via spectrophotometer. Western blot analysis was performed to investigate the effects of royal jelly on the protein expression levels of Bax, caspase-3, caspase-6, Bcl-2, NF-κB, COX-2, and Erk. It was also studied the effects of RJ on histopathological alterations in fluoride-induced damage to the rat brain. As a result, the Bcl-2, NF-κB, and COX-2 protein expression levels were increased in the fluoride-treated (50 and 100 mg/kg) groups but they were decreased significantly by RJ treatment in the brain tissue. Additionally, the protein expression of caspase-3, caspase-6, Bax, and Erk were decreased in fluoride-treated groups and they were significantly increased by RJ treatment compared to the un-treated rats. Our results suggested that RJ prevented fluoride-induced brain tissue damage through anti-antioxidant activities.

Discovery of New Heterocyclic/Benzofuran Hybrids as Potential Anti-Inflammatory Agents: Design, Synthesis, and Evaluation of the Inhibitory Activity of Their Related Inflammatory Factors Based on NF-κB and MAPK Signaling Pathways.

NF-κB and MAPK are classic inflammation signaling pathways which regulate inflammation signal transmission and induce the expression of many inflammatory factors. Based on the potent anti-inflammatory activity of benzofuran and its derivatives, several new heterocyclic/benzofuran hybrids were first designed and synthesized by molecular hybridization. Their structure was confirmed by 1H NMR, 13C NMR, HRMS or X-single crystal diffraction. The anti-inflammatory activity of these new compounds was screened by compounds; compound 5d exhibited an excellent inhibitory effect on the generation of NO (IC50 = 52.23 ± 0.97 µM), and low cytotoxicity (IC50 > 80 µM) against the RAW-264.7 cell lines. To further elucidate the possible anti-inflammatory mechanisms of compound 5d, the hallmark protein expressions of the NF-κB and MAPK pathways were studied in LPS-stimulated RAW264.7 cells. The results indicate that compound 5d not only significantly inhibits the phosphorylation levels of IKKα/IKKß, IKßα, P65, ERK, JNK and P38 in the classic MAPK/NF-κB signaling pathway in a dose-dependent manner, but also down-regulates the secretion of pro-inflammatory factors such as NO, COX-2, TNF-α and IL-6. Further, the in vivo anti-inflammatory activity of compound 5d indicated that it could regulate the involvement of neutrophils, leukocytes and lymphocytes in inflammation processes, and reduce the expression of IL-1ß, TNF-α and IL-6 in serum and tissues. These results strongly suggest that the piperazine/benzofuran hybrid 5d has a good potential for developing an anti-inflammatory lead compound, and the anti-inflammatory mechanism might be related to the NF-κB and MAPK signaling pathways.

Daidzein phosphorylates and activates 5-lipoxygenase via the MEK/ERK pathway: a mechanism for inducing the production of 5-lipoxygenase metabolite that inhibit influenza virus intracellular replication.

We previously reported that the soy isoflavone daidzein (Dz) suppresses the intracellular replication of influenza virus and that arachidonic acid-derived oxidation product via lipid oxidase 5-lipoxygenase (5-LOX) is involved in its antiviral effect. The activation of 5-LOX by Dz triggers anti-influenza activity; however, the mechanism of activation of 5-LOX remains unclear. Therefore, in this study, we aimed to clarify the activation mechanism using human monocyte-derived THP-1 cells differentiated using phorbol 12-myristate 13-acetate. THP-1 cells expressed 5-LOX endogenously and Dz did not induce 5-LOX expression. However, 8 h after treatment with Dz, the amount of 5-hydroxyeicosatetraenoic acid (5-HETE), an arachidonic acid oxidation product via 5-LOX, increased significantly suggesting that the enzyme is activated regardless of changes in 5-LOX protein levels. Intracellular Ca2+ content, ATP concentration, 5-LOX protein phosphorylation, and 5-LOX intracellular localization are known 5-LOX activation factors. The intracellular Ca2+ and ATP concentrations were not affected by Dz treatment. The enzymatic activity of 5-LOX is regulated by the phosphorylation of three serine residues and four tyrosine residues. Pretreatment with inhibitors of each kinase revealed that Dz-induced 5-HETE production was suppressed by the MEK/ERK inhibitor. 5-LOX in which the Ser663 residue was phosphorylated was found to be increased in the nuclear fraction of Dz-treated THP-1 cells. Furthermore, immunocytochemistry showed that 5-LOX translocates to the nuclear envelope following Dz treatment. These results indicate that Dz activates 5-LOX by phosphorylating Ser663 via the MEK/ERK pathway. Thus, these results demonstrate that Dz exerts anti-influenza virus activity via the MEK/ERK signal transduction pathway.

ASK1 inhibitor NQDI­1 decreases oxidative stress and neuroapoptosis via the ASK1/p38 and JNK signaling pathway in early brain injury after subarachnoid hemorrhage in rats.

Oxidative stress and neuroapoptosis are key pathological processes after subarachnoid hemorrhage (SAH). The present study evaluated the anti­oxidation and anti­apoptotic neuroprotective effects of the apoptosis signal­regulating kinase 1 (ASK1) inhibitor ethyl­2,7­dioxo­2,7­dihydro­3H­naphtho(1,2,3­de)quinoline­1­carboxylate (NQDI­1) in early brain injury (EBI) following SAH in a rat model. A total of 191 rats were used and the SAH model was induced using monofilament perforation. Western blotting was subsequently used to detect the endogenous expression levels of proteins. Immunofluorescence was then used to confirm the nerve cellular localization of ASK1. Short­term neurological function was assessed using the modified Garcia scores and the beam balance test 24 h after SAH, whereas long­term neurological function was assessed using the rotarod test and the Morris water maze test. Apoptosis of neurons was assessed by TUNEL staining and oxidative stress was assessed by dihydroethidium staining 24 h after SAH. The protein expression levels of phosphorylated (p­)ASK1 and ASK1 rose following SAH. NQDI­1 was intracerebroventricularly injected 1 h after SAH and demonstrated significant improvements in both short and long­term neurological function and significantly reduced oxidative stress and neuronal apoptosis. Injection of NQDI­1 caused a significant decrease in protein expression levels of p­ASK1, p­p38, p­JNK, 4 hydroxynonenal, and Bax and significantly increased the protein expression levels of heme oxygenase 1 and Bcl­2. The use of the p38 inhibitor BMS­582949 or the JNK inhibitor SP600125 led to significant decreases in the protein expression levels of p­p38 or p­JNK, respectively, and a significant reduction in oxidative stress and neuronal apoptosis; however, these inhibitors did not demonstrate an effect on p­ASK1 or ASK1 protein expression levels. In conclusion, treatment with NQDI­1 improved neurological function and decreased oxidative stress and neuronal apoptosis in EBI following SAH in rats, possibly via inhibition of ASK1 phosphorylation and the ASK1/p38 and JNK signaling pathway. NQDI­1 may be considered a potential agent for the treatment of patients with SAH.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

PURPOSE: Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrent MYC and BCL2 rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. METHODS: Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. RESULTS: In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. CONCLUSION: In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways.

Bladder cancer is a common global cancer with a high percentage of metastases and high mortality rate. Thus, it is necessary to identify new biomarkers that can be helpful in diagnosis. Pyruvate dehydrogenase kinase 4 (PDK4) belongs to the PDK family and plays an important role in glucose utilization in living organisms. In the present study, we evaluated the role of PDK4 in bladder cancer and its related protein changes. First, we observed elevated PDK4 expression in high-grade bladder cancers. To screen for changes in PDK4-related proteins in bladder cancer, we performed a comparative proteomic analysis using PDK4 knockdown cells. In bladder cancer cell lines, PDK4 silencing resulted in a lower rate of cell migration and invasion. In addition, a PDK4 knockdown xenograft model showed reduced bladder cancer growth in nude mice. Based on our results, PDK4 plays a critical role in the metastasis and growth of bladder cancer cells through changes in ERK, SRC, and JNK.

Resveratrol Nanoparticles Suppresses Migration and Invasion of Renal Cell Carcinoma Cells by Inhibiting Matrix Metalloproteinase 2 Expression and Extracellular Signal-Regulated Kinase Pathway.

The aim of this study was to examine the impact of Resveratrol nanoparticles on migration/invasion capacity of renal cell carcinoma (RCC) cells and its mechanism. Human RCC cells were exposed to dimethyl sulfoxide or gradient concentrations of Resveratrol nanoparticles respectively, and U0126 were also added in some experiments. We examined renal cell viability by MTT assay, and wound healing test and Transwell assays were used detect invasion and migration capability of RCC cells. We used Western blotting assay to analyze the protein levels in extracellular signal-regulated kinase (ERK) signaling. We also detected the enzymatic capacity of matrix metalloproteinase 2 (MMP-2) in cells by gelatin enzymatic profiling. Resveratrol nanoparticles treatment significantly suppressed cell viability to migrate and invade RCC cells in a dose-dependent manner. Also, notably were reduced MMP-2 activity and expression, and elevated TIMP-2 level were observed in RCC cells exposed with Resveratrol nanoparticles. Further, Resveratrol nanoparticles treatment significantly decreased only the expression of p-ERK1/2, but not p-p38 and p-JNK. Moreover, U0126, which is the ERK inhibitor, exerted similar role as Resveratrol nanoparticles did. Of note was that, combined use of U0126 and Resveratrol nanoparticles displayed a more intense suppression of MMP-2 activity and expression, and also the viability to migrate and invade the RCC cells, compared with Resveratrol nanoparticles treatment alone. The Resveratrol nanoparticles inhibited RCC cells migration and invasion by regulating MMP2 expression and ERK pathways.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co-administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle-treated mice. Compared to vehicle-treated mice, ERKi-treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho-Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi-treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co-administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway.

BACKGROUND: High glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibits abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic differentiation under high glucose and the underlying mechanisms remain unknown. METHODS: Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways. RESULTS: We found that osteogenic differentiation of PDLSCs under high glucose was decreased, and metformin addition enhanced this capacity of differentiation. Furthermore, the results of RNA-seq analysis showed that natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. When the underlying pathways involved were investigated, we found that upregulation of NPR3 can compromise the metformin-enhanced PDLSC osteogenic differentiation and activate the MAPK pathway (especially the p38 MAPK and Erk1/2 pathway), and that inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs under high glucose. CONCLUSIONS: The present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.

Nilotinib modulates LPS-induced cognitive impairment and neuroinflammatory responses by regulating P38/STAT3 signaling.

BACKGROUND: In chronic myelogenous leukemia, reciprocal translocation between chromosome 9 and chromosome 22 generates a chimeric protein, Bcr-Abl, that leads to hyperactivity of tyrosine kinase-linked signaling transduction. The therapeutic agent nilotinib inhibits Bcr-Abl/DDR1 and can cross the blood-brain barrier, but its potential impact on neuroinflammatory responses and cognitive function has not been studied in detail. METHODS: The effects of nilotinib in vitro and in vivo were assessed by a combination of RT-PCR, real-time PCR, western blotting, ELISA, immunostaining, and/or subcellular fractionation. In the in vitro experiments, the effects of 200 ng/mL LPS or PBS on BV2 microglial cells, primary microglia or primary astrocytes pre- or post-treated with 5 µM nilotinib or vehicle were evaluated. The in vivo experiments involved wild-type mice administered a 7-day course of daily injections with 20 mg/kg nilotinib (i.p.) or vehicle before injection with 10 mg/kg LPS (i.p.) or PBS. RESULTS: In BV2 microglial cells, pre- and post-treatment with nilotinib altered LPS-induced proinflammatory/anti-inflammatory cytokine mRNA levels by suppressing AKT/P38/SOD2 signaling. Nilotinib treatment also significantly downregulated LPS-stimulated proinflammatory cytokine levels in primary microglia and primary astrocytes by altering P38/STAT3 signaling. Experiments in wild-type mice showed that nilotinib administration affected LPS-mediated microglial/astroglial activation in a brain region-specific manner in vivo. In addition, nilotinib significantly reduced proinflammatory cytokine IL-1ß, IL-6 and COX-2 levels and P38/STAT3 signaling in the brain in LPS-treated wild-type mice. Importantly, nilotinib treatment rescued LPS-mediated spatial working memory impairment and cortical dendritic spine number in wild-type mice. CONCLUSIONS: Our results indicate that nilotinib can modulate neuroinflammatory responses and cognitive function in LPS-stimulated wild-type mice.